In cell biology, for example, neurons presently are examined with conventional light microscopes. The cells inclusive of their dendrites are often larger than the field size, determined substantially by the aperture of the objective, of the microscope. A larger field can be observed with low-magnification objectives, but usually the requisite resolution cannot be obtained therewith.
An attempt is made to solve this problem by manually delineating the image of the specimen piece by piece, successive different parts of the specimen being shifted into the observation field of the conventional microscope and manually focused in each case.
It often takes hours to obtain an overall view of a specimen that is large as compared to the field size, for example of a neuron with its long dendrites; this is thus very time-consuming and consequently cost-intensive. In addition, the lifetime of suitably prepared cells is sometimes only in the range of one to two hours, so that certain experiments cannot be performed at all.
It is possible to acquire the image of a specimen more quickly and accurately using a suitable scanning device. The scanning device can also be constituted by a scanning microscope. In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane with the aid of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another so that one mirror deflects in the X and the other in the Y direction. Tilting of the mirrors is brought about, for example, using galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. Ideally, the track of the scanning light beam describes a meander on or in the specimen (scan one line in the X direction at constant Y position; then stop X scanning and slew by Y displacement to the next line to be scanned; then, at constant Y position, scan that line in the negative X direction, etc.).
For fundamental reasons, however, the scan region of a scanning microscope is no larger than that of a conventional light microscope that is equipped with comparable optical elements (objective, tube lens, etc.). A scanning microscope nevertheless offers the advantage of storing the image data of a scan region and scanning the adjacent regions in subsequent steps. In this context, the specimen is successively displaced in meander fashion using a displacement stage. With the aid of suitable software, the image data that correspond to the individual adjacent scan regions are then linked to one another so they can be assembled into one overall image. Here again, it is desirable to arrange the scan region serially in meander fashion, in order to eliminate redundancies. To carry out this method, the specimen table is usually moved automatically in computer-controlled fashion until the entire specimen plane has been scanned. A procedure of this kind is described in W. Zuschratter, T. Steffen, K. Braun, A. Herzog, B. Michaelis, and H. Scheich (1998), “Acquisition of multiple image stacks with a confocal laser scanning microscope” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing, V. Carol, J. Cogswell, J. A. Conchello, J. M. Lerner, T. Lu, T. Wilson (eds.), Proceedings of SPIE Vol. 3261, pp. 177-186.
When linking the image data that correspond to the individual adjacent scan regions, the software has the task of taking into account image distortions, for example pincushion distortions. Mere “sequential copying” of the relevant image data is generally not sufficient, and yields very poor results. An angular error between the displacement directions of the specimen stage and the scanning axes (“crabbing error”) is very particularly troublesome in this context.
In most cases the specimens that are to be scanned are not flat but rather are three-dimensional objects, which greatly complicates documentation in particular by manual delineation, and sometimes yields unsatisfactory results.
It is first of all conceivable to use a confocal scanning microscope, which is inherently capable of scanning a specimen three-dimensionally. In confocal scanning microscopy, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the “excitation stop”), a beam splitter, a beam deflection device for beam control, an optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused onto the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop; a point datum is thus obtained that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by image acquisition in layers; a scan field that is defined by the focusing optical system is displaced correspondingly by way of a relative motion between the specimen stage and the focusing optical system. The result of this relative motion is that the scan field is moved in the Z direction through the specimen being examined.
Since the horizontal cross section of the scan volume of a confocal scanning microscope is again, when comparable optics are used, in principle no larger than the field size of a conventional light microscope, the fundamental difficulty of scanning specimens that are larger than the scan field size is not overcome simply by using a confocal scanning microscope.
In confocal scanning microscopy it is also possible and usual to obtain, by meander-shaped displacement of the specimen, image information from the specimen that is larger than the scan field being used. Scanning is performed in different layers for each scan field, and the image data obtained are then linked into one coherent image. The image data encompass the entire space containing the specimen. It is easy to imagine that in the case of specimens branching off extensively in three dimensions, a great deal of unnecessary space containing no image data is also scanned. Time is wasted in scanning, since regions which contain no specimen image data are scanned unnecessarily.
Just like the manual delineation method, the scanning-microscopy methods described above are very time-consuming. In addition, the results of the image data linkage are not satisfactory because aberrations are not taken into account.